Two major forms of Phospholipase D(PLD) activity were separated from rat brain membranes by chromatography on an HPLC column. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADP-ribosylation factor (ARF) and GTPgammaS, required the presence of phosphatidylinositol 4,5- bisphosphate (PIP2) in the phosphatidylcholine substrate for demonstration of activity. Both sodium oleate- and ARF-dependent activities catalyzed transphosphatidylation, thus, identifying them as PLDs. Oleate-dependent activity was unaffected by ARF, GTPgammaS, or PIP2.